This project will investigate how drug compounds interact with dopamine transporter (DAT) utilizing an integrated approach combining biochemical and computational methodologies. Biochemical analyses will use novel photoaffinity inhibitors that irreversibly attach to the transporter. These studies will determine points of contact between the drug and DAT and the molecular orientation of the ligand in the binding site. In parallel, we will carry out computational studies to build comparative models of DAT based on homology to a competitor-bound crystal structure from a related bacterial transporter, LeuT. The comparative models will be used in conjunction with the biochemical results to computationally dock the photoaffinity ligands into DAT. Recent advancements in computational protein folding have legitimized its use in modeling integral membrane proteins. Docked structures consistent with current and ongoing biochemical data will be further refined using molecular dynamics. The results obtained from biochemical and computational analyses will lead to hypotheses that will be experimentally tested using site-directed mutagenesis, cysteine-scanning accessibility, and a library of DAT antagonist analogs to evaluate the molecular predictions. The integration of findings from these approaches will provide significant information related to the structure of the DAT active site and how antagonists exert effects on transport. PUBLIC HEALTH RELEVANCE: The dopamine transporter is a major target of several drugs of abuse and has been linked to addiction and drug seeking behaviors, and as such is a highly clinically relevant protein. However, we still do not understand many of the details regarding the basis of drug recognition at DAT, or how various DAT drugs induce particular behavioral outcomes. Understanding the molecular basis of these properties could lead to important advances in clinical targeting of DAT as well as the related norepinephrine and serotonin transporters, all which play critical roles in our neurological and psychological health.